Chronic morphine exposure leads to stable changes in gene expression in the brain reward, pain, and learning and memory circuits, resulting in neuroadaptations that underlie drug tolerance, physical dependence, and withdrawal (symptoms associated with both physical dependence and motivational withdrawal). Since binding of nuclear proteins to the regulatory regions of a gene is one of the fundamental steps to regulate transcription of the gene, unbiased and quantitative analysis of the genome-wide protein-DNA interactions responding to chronic morphine and morphine withdrawal will be one of the critical steps to unravel the complex molecular mechanism of morphine dependence. The primary objective of this cutting-edge basic research awards (CEBRA) R21 application is to develop a novel tandem ChIP-Seq approach by combining the cutting-edge tandem ChIP (Re-ChIP, or Sequential ChIP) and next generation DNA sequencing methods to identify and analyze genome-wide protein-DNA interactions responding to chronic morphine and withdrawal. We will establish this method to analyze genome-wide CBP/p300-NF-:B/p65-DNA interactions in mu and delta opioid receptor-expressing cells after acute and chronic morphine (Specific Aim 1) and to apply the tandem ChIP-Seq method to studying genome-wide co-occupancy of CBP/p300 and NF-kB/p65 in the striatum of rats chronically exposed to morphine and after naloxone-precipitated withdrawal (Specific Aim 2). Successful completion of these two aims together will provide the proof of principle to use the tandem ChIP-Seq method to identify and analyze cis-elements, enhancers, and drug-regulated genes, including non-coding RNA genes in drug abuse and addiction models, and may eventually identify new molecular targets for prevention and treatment of drug dependence. PUBLIC HEALTH RELEVANCE: The proposed study is to develop a new genome-wide analysis approach for opioid dependence research. Successful completion of this study will provide a new research method and the first time information on genome-wide protein-DNA interactions differentially responding to chronic morphine exposure and naloxone-precipitated withdrawal in the rat model. The new information may eventually reveal new molecular targets for prevention and treatment of opioid dependence.